QIAseq FX Single Cell RNA Library Kit

Single cell RNA-seq libraries that provide a deeper view of the transcriptome

Higher diversity libraries detect more transcripts, including mRNA and lncRNA

PCR-free protocol reduces bias and provides great reproducibility

Sequencing ready libraries from isolated single cells in just 5.5 hours

The QIAseq FX Single Cell RNA Library kit is an end-to-end library preparation solution for RNA-seq from single cells or low amounts of RNA. The kit includes all reagents required for cell lysis, reverse transcription, cDNA amplification and PCR-free NGS library preparation.It produces high-quality NGS libraries and generates a tube of excess amplified cDNA that can be stored for follow-up experiments. The kit is ideally suited for transcript discovery and differential expression from single eukaryotic cells and RNA-seq from limited samples including viral RNA.

The QIAseq FX Single Cell RNA Library Kit detects a greater number of transcripts at the same sequencing depth. To account for cell-to-cell differences in transcript abundance, libraries were produced from 100 pg of reference RNA from PBMCs. After sequencing, quality control and mapping, annotated transcripts with >1 TPM were quantified from either the full dataset or rarified sub-fractions. Saturation curves are from different sample preparation methods. Each point on the curve was generated by randomly selecting a number of raw reads from each sample library and then using the same alignment pipeline to call genes with mean TPM>1.|Single Cell RNA-Libraries from PBMCs were generated using QIAseq FX Single Cell RNA Library kit und a Kit from Supplier C/I. Libraries were sequenced on Illumina NextSeq. Plotted is the % of duplicates, that was obtained from the Fast QC report of the sequenced libraries.|Single Cell RNA-Libraries from PBMCs and HeLa Cells were generated using QIAseq FX Single Cell RNA Library kit und a kit from Supplier C/I. Plotted are the percentage of reads that map to linc RNA detected in PBMC and HeLa preparations. QIAseq detects a significantly higher percentage of long regulatory RNAs compared to Supplier C/I.|Single cell libraries were prepared from PBMCs or toRNA from PBMCs using the QIAseq FX Single Cell RNA Library Kit and sequenced on NextSeq. Plotted is the % of reads that map to different RNA biotypes.| |4 Individual HeLa cells were isolated from the same cell culture and libraries were prepared with either the QIAseq FX Single Cell RNA Library Kit or a competing workflow. After sequencing to equal depth, quality control, alignment, and TPKM calculation, pairwise comparisons of the number of common transcripts detected with >1 TPKM divided by sum of all transcripts in both preps were made between all tested cells. The graphs represent mean of 6 pairwise comparisons with SD.|

The kit detects a greater number of transcripts than competing methods at any sequencing depth. In some single-cell RNA-seq workflows PCR is used extensively for both cDNA amplification and to amplify the minute amounts of library produced. This has a negative effect on library diversity from the loss of low-abundance transcripts due to both stochastic effects and PCR-bias during cDNA amplification and the generation of PCR duplicates during library amplification. Additionally, these PCR systems often introduce GC-bias and length-bias, which can make the sequencing of GC-rich or long transcripts difficult. In contrast, the QIAseq FX Single Cell RNA Library Kit relies on a highly-efficient MDA reaction to amplify cDNA, and generates enough material that library amplification is not necessary, delivering a completely PCR-free workflow. This eliminates PCR duplicates and maximizes the number of transcripts detected, including long transcripts such as lncRNAs.

Principle

This kit relies on three key technologies to enable PCR-free RNA-seq from single cells. After cell lysis, gDNA degradation and reverse transcription, the cDNA is ligated to generate a long template that can be amplified in a multiple displacement annealing reaction. This is performed with a high-fidelity, ultra-pure version of the phi29 polymerase, and generates µg amounts of long cDNA. This long cDNA is then fragmented with the random FX technology into short inserts suitable for sequencing. Finally, a highly optimized and efficient single-tube library preparation packages these inserts into NGS libraries which can be sequenced directly, without the need for library amplification and additional PCR-bias. Taken together, the high-yield MDA, random and unbiased FX fragmentation, and efficient single-tube library construction deliver highly-complex PCR-free RNA-seq libraries from single cells in only 5.5 hours.

Procedure

The QIAseq FX Single Cell RNA Library Kit is a complete cell-to-library solution that generates RNA-seq libraries from single eukaryotic cells or from pg-levels of purified RNA. The kit contains reagents for the efficient lysis of isolated single cells from common cell-sorting or cell-isolation platforms. After lysis, genomic DNA is degraded and reverse transcription is performed. This reaction can be primed from either an included oligo-dT primer in order to reduce the number of reads arising from ribosomal RNA and to enrich for polyadenylated RNAs, or from random primers, for example when sequencing viral RNA genomes from limited samples. After reverse transcription and second strand synthesis, a ligation step generates a long, double-stranded cDNA template which is amplified via multiple displacement annealing using a specially engineered, ultra-high fidelity phi29 polymerase. This generates µg amounts of amplified cDNA, some of which can be stored for later use or confirmatory testing. Amplified cDNA is fragmented with a highly random, enzymatic fragmentation step, which generates inserts compatible with common sequencing read lengths, and insert size can be varied according to individual preferences. No exact quantification of the cDNA is necessary prior to fragmentation, as both the amplification process and fragmentation reaction are extremely robust. Insert DNA is end-polished and subjected to a highly efficient ligation reaction with the included barcoded adapters, which can be purified by standard methods prior to quantification and sequencing. Amplification of the library with PCR is not necessary, so the production of PCR duplicates is avoided and library diversity remains high.